Studies On The Directed Differentation Of CD4~+T Cells By Enterococcus Facium HDRsEf1 Or Its Extracellulae Polysaccharides In The Cross-talk System

Enterococcus facium HDRsEf1 strain is a high-quality probiotic strain obtained from the rectum content of the local high-quality“Tongcheng pig”,which has the advantages of reducing the diarrhea rate of piglets,improving the growth performance of piglets and enhancing the intestinal barrier.It has been found that HDRsEf1 can protect the integrity of IPEC-J2 cells and reduce the secretion of IL-8 in IPEC-J2 cells.The anti-inflammatory effect of HDRsEf1 was related to its extracellular polysaccharides called EPS,the necessary evidences of the mechanism of HDRsEf1 to inhibit inflammation and maintain intestinal homeostasis were provided in previous researchs.The results of anti-inflammatory effects of HDRsEf1 have been derived from intestinal epithelial cell lines of pig in vitro.The simple model of intestinal epithelial cells was used to simulate the true intestinal of animals in a certain extent.Therefore,in this study,Epithelial cells and the DC,CD4~+T cells in a core of the immune status under epithelial cells were selected to constructe the co-culture models to simulate the cross-talk of intestinal cells,with the main line that DCs directly or indirectly stimulated by HDRsEf1 induced directional differentiation of CD4~+T cells to clarify the effects of Enterococcus facium HDRsEf1 and its extracellular polysaccharides on inhibiting inflammatory.In order to visualize the possible role of intestinal epithelial cells and the interaction between intestinal epithelial cells and DCs,the DC stimulated by probiotic in co-cultured cell model was designed in three different forms,called the Direct group(direct stimulation of BMDC with HDRsEf1 or EPS),Indirect group(treated BMDC with supernatants of MODE-K stimulated with E.faecium HDRsEfl or EPS),Transwell group(MODE-K located in the upper chamber,BMDC in the bottom chamber,with MODE-K stimulated with HDRs Efl or EPS).The main research contents and results are as follows:(1)Through tests of transmembrane resistance(TEER)and cytotoxicity on MODE-K sitimulated with HDRsEf1 or EPS,The 1×107 CFU HDRsEf1?25 ?g/m L APS and 25 ?g/m L ? 50 ?g/m L NPS were determined to stimulate BMDC.BMDC was separated and induced from mouse bone marrow,and the 5th day of BMDC in immature state was choosed for subsequent research;The na?ve CD4~+T cells were sorted from the mouse spleen by immunomagnetic bead sorting with 99% purity,which laid the foundation for the co-cultured cell model.(2)Effect of HDRsEf1 on maturation of BMDC and its cytokines.HDRsEf1 promoted the maturation of BMDC in Direct group,while HDRsEf1 inhibited thematuration of BMDC in indirect group and Transwell group.HDRsEf1 significantly increased the secretion of pro-inflammatory cytokines IL-1? and TNF-? of BMDC in direct group,HDRsEf1 inhibited the secretion of pro-inflammatory cytokines IL-1? and up-regulated the secretion of IL-10 and TGF-? in indirect group and Transwell group,which indicated that HDRsEf1 can directly exert anti-inflammatory effects through DC in the intestine.(3)Effect of directed differentiation of CD4~+T cells on BMDC stimulated by HDRsEf1.HDRsEf1 promoted the differentiation of CD4~+T cells into Th1 and Th2 types,and Th1 type predominated reaching to 30.4% in the direct group;HDRsEf1 did not promote the differentiation of CD4~+T cells to Th1,Th2 and Th17 types in the indirect group;HDRsEf1 significantly inhibited the differentiation of CD4~+T cells into Th1 and Th17 subtypes,and promoted differentiation of CD4~+T cells into Th2 type reaching to16.6% in Transwell group.It was suggested that HDRsEf1 can induce CD4~+T cell directed differentiation to indirectly exert anti-inflammatory effects in the intestine.(4)Effect of HDRsEf1 on maturation of BMDC and its cytokines.25?g/m L APS and50?g/m L NPS promoted the maturation of BMDC,upregulated expression of IFN-?,IL-12,and TGF-? m RNA and secretion of pro-inflammatory factors IL-1?,IL-6,TNF-?in direct group,while 25?g/m L APS and 50?g/m L NPS both inhibited the maturation of BMDC,inhibited the expression of IL-12,IFN-? m RNA and inhibited the secretion of IL-1? in indirect group.25?g/m L APS and 25?g/m L NPS significantly promoted the maturation of BMDC and upregulated the secretion of pro-inflammatory cytokines in direct group.APS and NPS both inhibited the expression of surface molecules of BMDC and significantly inhibited the secretion of inflammatory cytokines in indirect and Transwell groups.It was suggested that the EPS of HDRsEf1 can inhibit the maturation of DC and directly exert the anti-inflammatory effect through DC in the intestine.(5)The effect of differentiation of CD4~+T cells on BMDC stimulated by EPS from Enterococcus faecium HDRsEf1.The results showed that APS and NPS both inhibited the proliferation and differentiation of CD4~+T cells.In the immunoregulation of probiotic HDRsEf1 on the intestine and even the whole body,intestinal epithelial cells may interact with the underlying DCs through their secreted cytokines,which play an important role in regulating immune homeostasis.